The rho genes comprise an evolutionarily conserved family with significant homology to the ras oncogene family. The implied structural similarity of rho and ras proteins suggests that the two families evolved from a common ancestral gene, and that the proteins share common biochemical functions. In the yeast Saccharomyces cerevisiae, similar mutations in either rho or ras genes prevent the normal sporulation response to nutrient limitation. These and other observations suggest that rho and ras proteins function in signal transduction processes. Despite the conservation between the rho and ras families, preliminarily experiments have shown that rho and ras function in distinct biochemical pathways. Therefore, rho and ras proteins appear to utilize common functions to achieve different physiological effects. The overall goal of this project is to determine the function of rho proteins and to evaluate the functional similarities and differences between the rho and ras gene families. The yeast gene RHO1 will be the initial subject of the studies, due to preliminary results indicating its involvement in cellular response to the environment. Two complementary approaches will be used simultaneously to investigate RHO1 function. First, the protein will be purified and biochemically characterized in vitro. The eventual aim of these studies is the reconstruction in vitro of a functional RHO1 signal transduction system. The second approach will use the techniques available for isolating specific regions of the yeast genome, engineering this material to desired specifications, and reinserting the altered DNA into the chromosome. This approach will seek to construct an in vivo system for assay of RHO1 function, through the use of conditional-lethal alleles. The genetic approach will also investigate biochemical pathways with which RHO1 interacts, through the identification and isolation of second-site suppressor mutations that modulate RHO1 function. These genetic experiments will complement the biochemical studies by indicating specific RHO1 activities to be tested in vitro. rho genes will be examined for function in heterologous systems, thus revealing whether the family of proteins has been functionally conserved in evolution. In addition to this general investigation of rho activity, the functional relationship of specific regions of rho and ras proteins will be examined by construction of chimeric genes containing domains from both divisions of the ras-rho superfamily.